This dissertation describes the development and application of an approach for creating multiphoton crosslinked protein microchambers to characterize bacterial group behaviors in small populations (~10¹ - 10⁵ cells). Porous protein cavities of desired size and geometry are made with sub-micrometer three-dimensional (3D) resolution using a dynamic mask-based multiphoton lithography (MPL) technique previously developed in the Shear Group. One aspect of this dissertation focuses on basic characterizations of properties of these materials key to their utility in studying entrapped bacteria. Studies are presented on the mass transport across microcavity walls (important for growth and signaling), and the temperature- and light-induced volume response (used to open/close microchamber apertures for cell entry/exit). Fabrication parameters are optimized to trap and manipulate small populations under in vitro conditions that are relevant to in vivo environments. The ability to culture bacteria at physiologic growth rates within protein microstructures has provided a unique platform to study the group behaviors of quorum sensing (QS) and antibiotic resistance in biologically relevant population sizes, a platform I have exploited to study group behaviors in the opportunistic pathogen, Pseudomonas aeruginosa. This work presents the first experimental evidence supporting the efficiency sensing QS model by showing that QS-dependent gene expression is affected by both the population size and density, as well the external flow rate in the surrounding environment. The onset of antibiotic resistance is observed in as few as ~150 P. aeruginosa cells, and is shown to increase with cell density. Lastly, the development of a gelatin-based MPL approach that is demonstrated in situ to create confined populations of non-motile cells, free-floating 3D cultures, nested colonies, and spatially patterned polymicrobial communities of P. aeruginosa and Staphylococcus aureus. / text

Fluorescence from endogenous molecules and exogenous contrast agents can provide morphological, spectral, and lifetime contrast that indicates disease state in epithelial tissues. Recently, nonlinear microscopy has emerged as a potential tool for the early detection, case-finding, and monitoring of epithelial cancers because it permits non-invasive, three-dimensional fluorescence imaging of subcellular features hundreds of microns deep. This dissertation explores the use of nonlinear microscopy for cancer diagnostics on two fronts: (1) we examine the fundamental limitations governing the maximum nonlinear imaging depth in epithelial tissues, and (2) we investigate the use of a new class of nonlinear contrast agent---plasmonic gold nanoparticles---for molecularly specific imaging of cancer cells. We built and optimized a nonlinear microscope for deep tissue imaging, and studied the image contrast as a function of imaging depth in ex-vivo human biopsies and tissue phantoms. With this system we demonstrated imaging down to 370 [mu]m deep in a human biopsy, which is significantly deeper than imaging depths achieved in comparable studies. We found that the large scattering coefficient and homogenous fluorophore distribution typical of epithelial tissues limit the maximum imaging depth to 3-5 mean free scattering lengths deep in conventional nonlinear microscopy. Beyond this imaging depth, the increasing contribution of out-of-focus emission limits the contrast to insufficient levels for diagnostic imaging. We support these observations with time-dependent Monte Carlo simulations. We exploited the intense interaction of gold nanoparticles with light, enhanced by surface plasmon resonance effects, to create extremely bright nonlinear contrast agents. These contrast agents proved to be several orders of magnitude brighter than the brightest organic fluorophores and at least one order of magnitude brighter than quantum dots. We targeted gold nanoparticles to a biomarker for carcinogenesis and demonstrated molecularly specific imaging of cancer cells. We demonstrated that unlike emission from traditional bandgap fluorophores, nonlinear luminescence from gold nanoparticles was weakly dependent on excitation pulse length for short pulse durations. This finding supports the hypothesis that nonlinear excitation in plasmonic nanoparticles involves sequential rather than simultaneous absorption of excitation photons. The remarkable brightness of gold nanoparticles makes them an attractive contrast agent for nonlinear diagnostics. / text

Dynamic materials which can sense changes in their surroundings and subsequently respond or adapt by autonomously altering their functionality, surface chemistry, transparency, color, wetting behavior, adhesiveness, shape, etc. are primed to be integral components of future "smart" technologies. However, such systems can be quite complex and often require intricate coordination between both chemical and mechanical inputs/outputs as well as the combination of multiple materials working cooperatively to achieve the proper functionality. It is critical to not only understand the fundamental behaviors of existing dynamic chemo-mechanical systems, but also to apply that knowledge and explore new avenues for design of novel materials platforms which could provide a basis for future adaptive technologies. Part 1 explores the use of environmentally-sensitive hydrogels, either alone or within arrays of high-aspect-ratio nano/microstructures, as chemo-mechanical actuators. Chapters 1 through 7 describe a bio-inspired approach to the design of hybrid actuating surfaces in which the volume-changing hydrogel acts as the “muscle” that reversibly actuates the microstructured "bone". In particular, the different actuation mechanisms arising from variations in how the hydrogel is integrated into the structure array, how chemical signals can be used to manipulate actuation parameters, and finally how such a system may be used for applications ranging from adaptive optics to manipulation of chemical reactions are described. Chapter 8 discusses the use of responsive hydrogel scaffolds as a means to mechanically compress cells and direct differentiation. Part II explores dynamic microsystems involving the integration of catalytic sites within intricately structured 3D microenvironments. Specifically, we explore a generalizable and straightforward route to fabricate microscale patterns of nanocrystalline platinum and palladium using multiphoton lithography. The catalytic, electrical, and electrochemical properties are characterized, and we demonstrate high resolution integration of catalysts within 3D-defined microenvironments to generate directed particle and fluid transport. / Chemistry and Chemical Biology

Cancer currently represents one of the greatest burdens on human health in the world, claiming in excess of 7 million lives a year worldwide. Advances in both our understanding of the disease as well as our ability to diagnose it before it has had a chance to metastasize will lead to a reduction in its burden on society. To these ends, optical imaging techniques are particularly attractive. The ability to resolve cellular details noninvasively is paramount to improved cancer detection and to research on diseased tissue and cells. Lanthanoid nanoparticles, a group of photoluminescent contrast agents developed within the last two to three decades, have numerous unique optical properties that enable their use in improved and novel optical techniques. They possess large Stokes and anti-Stokes shifts, sharp electronic transitions, long luminescence lifetimes, and exceptional photostability. For these reasons, they are a good choice for biomedical applications that benefit from low background fluorescence or long illumination times. The major goal of the research presented in this dissertation was to synthesize functional lanthanoid nanoparticles for optical imaging modalities, and to explore their potential uses in a variety of biomedical applications. To this end, the research can be broken up into three specific aims. The first aim was to successfully and reproducibly synthesize downconverting and upconverting lanthanoid nanoparticles, and to functionalize these nanoparticles for use in optical techniques that would aid in the research and diagnosis of cancer. The second aim was to conduct a thorough investigation of the optical properties of these nanoparticles, and the third aim was to explore the utility of these nanoparticles in a variety of biomedical applications. First, both downconverting and upconverting lanthanoid nanoparticles were synthesized using several different methods, resulting in nanoparticles of varying size and surface functionality. Novel methods were employed to improve the utility of these nanoparticles for specific applications, including the incorporation of a mixed surface ligand population in downconverting lanthanoid nanoparticles and the use of a biomimetic surface coating to render upconverting nanoparticles water dispersible. These coated particles were further functionalized by the addition of folic acid and an antibody for epidermal growth factor receptor, both of which bind to cell surface receptors overexpressed in a number of cancers. Second, the spectral properties of lanthanoid nanoparticles were explored in detail, with special attention paid to many of the unique optical properties of upconverting lanthanoid nanoparticles. This included the discovery of one such unique property, the excitation frequency dependent emission of NaYF₄ nanocrystals codoped with Yb³⁺ and Er³⁺. Third, lanthanoid nanoparticles were used as contrast agents in a number of biomedical applications, including the development of a homogenous assay based on diffusion enhanced luminescence resonance energy transfer, a wide-field luminescence lifetime microscope, and a super resolution microscope based on the aforementioned excitation frequency dependent emission of NaYF₄:Yb³⁺,Er³⁺ nanoparticles. Specific binding of functionalized upconverting lanthanoid nanoparticles was investigated with laser scanning multiphoton microscopy, and an image processing technique was developed to overcome the challenge of working with long lived luminescent contrast agents using this imaging modality.

The nanoscale manipulation of soft-matter, such as biological tissues, in its native environment has promising applications in medicine to correct for defects (eg. eye cataracts) or to destroy malignant regions (eg. cancerous tumours). To achieve this we need the ability to first image and then do precise ablation with sub-micron resolution with the same setup. For this purpose, we designed and built a multiphoton microscope and tested it on goldfish gills and bovine cells. We then studied light-matter interaction on a hard polymer (PMMA) because the nature of ablation of soft-matter in its native environment is complex and not well understood. Ablation and modification thresholds for successive laser shots were obtained. The ablation craters revealed 3D nanostructures and polarization dependent orientation. The interaction also induced localized porosity in PMMA that can be controlled.

<p>In this dissertation, we develop pump probe spectroscopy as a method to differentiate different chemical varieties of melanin, a common biopigment, and exploit these differences to improve the accuracy of melanoma diagnosis. This method gives insight into the chemical makeup and secondary structure of melanins. Pump probe spectroscopy is implemented in a scanning laser microscope as a form of multiphoton imaging, where it is used to image biopsies of human pigmented cutaneous lesions. Melanoma diagnosis is clinically challenging: the accuracy of visual inspection by dermatologists is highly variable and heavily weighted toward false positives. Even the current gold standard of biopsy results in varying diagnoses among pathologists. Using pump probe imaging, significant chemical and morphological changes were found between melanoma and melanocytic nevi, including increased eumelanin content, chemical heterogeneity and general pigmentation. Signal processing methods revealed further differences between melanoma and melanocytic nevi on the cellular scale. Pump probe imaging directly in H&E stained biopsy samples allows integration of this technique with existing histopathology protocols. High resolution imaging found chemical heterogeneity of melanin within pigmented cells. We show that oxyhemoglobin and deoxyhemoglobin may also be differentiated by pump probe imaging. Epi mode imaging of eumelanin, pheomelanin and microvasculature is demonstrated in vivo in human xenograft mouse models of melanoma.</p> / Dissertation

Thesis (Ph. D.)--McMaster University (Canada), 1990. / Source: Dissertation Abstracts International, Volume: 62-13, Section: A, page: 0000.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 113-123).

Thesis (PH. D.)--Michigan State University. Chemistry, 2008. / Title from PDF t.p. (viewed on Sept. 8, 2009) Includes bibliographical references (p. 134-148). Also issued in print.

Thesis (Ph. D.)--Duke University, 2007. / Includes bibliographical references.